Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 – 30 mg of pooled cryosections and measurements are directly compared across applications.
With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 μm sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.
The goal of this technique is to make multiple experimental analyses by different modalities, such as histology and gene expression, accessible from a single small skeletal muscle source tissue. Microscopy applications are the most sensitive to sample preservation methods, which must be carefully controlled to limit the formation of ice crystal artifacts during cryopreservation. Thus, method development is based on the tibialis anterior (TA) muscle frozen partially covered with embedding resin in a -140 °C liquid nitrogen-cooled 2-methylbutane bath as the source material for both immunofluorescence microscopy and gene expression analyses.
The need to use the same source material for diverse technical approaches is particularly important for intramuscular injection-based experiments where the left and right muscles represent different conditions, one experimental and one control. For example, in muscle regeneration studies, one muscle is injected with a toxin to cause widespread tissue damage while the contralateral muscle serves as a vehicle-injected control1. Similarly, gene therapy studies for muscle disorders typically begin with validation of the gene therapy vector by intramuscular injection to be compared with empty vector, unrelated vector or vehicle control on the contralateral side2. Therefore, it is not possible to source each TA muscle to a different application.
Common strategies to deal with this issue are: i) to use a different muscle group for each application, ii) to use additional mice, or iii) to cut off a piece of the muscle for each application. However, substantial differences between muscle groups make it difficult to compare data from separate applications, and additional animals increase expense and are poorly justified if other alternatives exist. Dividing the muscle after dissection to source different applications is the best option in many cases. However, the muscle pieces are often too small to use pulverization under liquid nitrogen or mechanical grinding techniques for homogenization2-5. As muscle is a highly structural tissue packed with extracellular matrix and contractile proteins, inadequate mechanical homogenization leads to a low yield of subsequent DNA, RNA or protein. The method detailed here allows small quantities of tissue from one source muscle for use in multiple applications, and the inclusion of cryosectioning and needle trituration improves mechanical homogenization for better RNA yield.
All animal procedures were approved by the University of Georgia Institutional Animal Care and Use Committee under animal use protocol A2013 07-016 (Beedle).
1. Cryopreservation of Unfixed Skeletal Muscle
2. Collect Cryosections for Histology and RNA Applications
3. RNA Isolation from Pooled Cryosections
4. Histological Analysis by Immunofluorescent Staining of Muscle Cryosections
Muscle cryosection RNA is high in quality and provides sufficient yield for most applications
Analyses of sixteen skeletal muscle RNA preparations are shown in Table 1 using 19.4 to 41 mg of pooled tibialis anterior (TA) muscle from 8 control mice. Both left (L) and right (R) TA muscles were prepared in regeneration experiments with muscles collected 3 days after longitudinal intramuscular injection of 25 μl of saline or 10 μM cardiotoxin to cause muscle injury using methods previously reported1. As shown in Table 1, the A260/280 ratios for muscle cryosection RNA are typically close to 2 or higher in these representative samples. As "pure" RNA is considered to have A260/280 of 2.0 and A260/280 of 1.8 to 2.2, the purity of the cryosection RNA samples is excellent9. Total RNA recovery was typically over 10 μg per sample with yields of 0.18 to over 1 μg of total RNA per mg of starting tissue, providing sufficient material for most downstream applications. Notably, RNA concentration, total RNA extracted, and RNA yield per mg of starting tissue from TA muscles 3 days post-toxin injury was significantly higher than from saline-injected TAs. This suggests that there is improved homogenization when muscle structure is disrupted by extensive injury and/or that there is an increase in gene transcription and/or RNA stability in 3 day regenerating muscle. The persistence of RNA quality was assessed by simple 1x TAE/1.5% agarose gel electrophoresis of 1 μl of muscle cryosection RNA after samples were stored at -20 °C for 18 months. Prominent 18S and 28S rRNA bands are still evident in samples demonstrating high RNA quality, even under suboptimal storage conditions (Figure 1A).
Muscle cryosection RNA supports downstream applications
One microgram of RNA per pooled cryosection sample was treated with DNase and reverse transcribed from oligo dT primers. Following RNase treatment, the total volume of the reverse transcribed cDNA was 30 μl. Simple non-quantitative PCR with excess amplification was run to confirm the viability of the cDNA. Myogenic regulatory factor 4 (Mrf4), a transcription factor upregulated with muscle differentiation, was amplified using previously reported mouse primers, sense 5'-CTACATTGAGCGTCTACAGGACC and antisense 5'-CTGAAGACTGCTGGAGGCTG10, from 2 μl of template using standard PCR. There was robust, specific amplification of the 234 bp Mrf4 fragment from both left and right TA cDNA samples, but not RT- (RNA included, but no reverse transcriptase), RT ddH2O (reverse transcription with no RNA template), or ddH2O PCR controls (Figure 1B). The same samples were run in triplicate for Mrf4 and mOaz1 reference control4 quantification using relative amplification and passive fluorescence reference on a real-time PCR system. Mrf4 was expressed at 0.097-fold in the toxin-injected right TA compared to the saline-injected left TA, calculated by the ΔΔCt method4. This is similar to previous reports of low Mrf4 expression 3 days after toxin injury due to a loss of mature muscle fibers11. To compare the consistency of cryosection RNAs for quantitative PCR, Ct values were compared for the mOaz1 reference gene. From six samples, mOaz1 transcript was detected with an average Ct of 17.242 ± 1.483 s.d., whereas the average Ct was 36.332 ± 3.61 s.d. in RT- control samples (n = 4). The tight grouping of mOaz1 Ct signals across samples suggests that RNA isolated from TA muscle cryosections performs as expected in downstream RNA expression analyses.
Histological assessment of adjacent cryosections.
Examples of indirect immunofluorescence staining of 7 μm tibialis anterior muscle sections from littermate control mice 3 days after toxin injection are shown to detect regenerating fibers (Figure 2). Both sections were collected from muscle less than 150 μm from the region to be used for RNA analysis at a depth of 4.5 to 4.6 mm from the proximal muscle surface. Embryonic myosin heavy chain (eMHC, red) detects regenerating fibers, collagen VI (ColVI, green) outlines the muscle fibers, and DAPI stains nuclei blue, according to protocol 4. Regions with concentrated nuclear infiltrate (blue signal) indicate sites of toxin injury, as evident by activation of eMHC positive newly regenerating muscle cells. Whole section maps like this example are used for quantification of regeneration by calculating the proportion of embryonic myosin heavy chain-expressing fibers (red, newly regenerated) or centrally nucleated fibers as reported previously1. Notably, there is surprisingly little damage in the TA muscle in Figure 2A. While there is variation between injections, typical toxin injections affect a much bigger proportion of the muscle compartment1, as shown in Figure 2B. Therefore, this histological analysis suggests that the toxin injury in Figure 2A was minimal and provides an important tool to interpret gene expression data from the contiguous muscle cryosection RNA sample. If the whole muscle had been used for RNA preparation by standard grinding methods, the unexpectedly small injury area of this sample would make it an outlier that would skew downstream analyses. Instead, pairing histological quantification from the same muscle allows direct measurement of the extent of the injury from adjacent sections. This enables the use of inclusion/exclusion criteria to ensure that all samples included in downstream RNA analyses meet a minimum injury threshold or the normalization of RNA analyses according to injury size.
Figure 1: Quality Assessment of RNA from Pooled Muscle Cryosections. A) 18S and 28S ribosomal RNA bands are prominent in RNA from pooled muscle cryosections 18 months after the RNA preparation. B) Non-quantitative PCR for Mrf4 following reverse transcription. 200 and 300 bp bands of a DNA ladder are indicated. RT-, reverse transcription reaction with RNA template but no reverse transcriptase. RT-H2O, reverse transcription with ddH2O, no RNA template. H2O, no template PCR control with ddH2O. In these experiments, toxin was injected into the right tibialis anterior (RTA) and saline was injected into the left (LTA). Please click here to view a larger version of this figure.
Figure 2: Sample Histological Maps for Muscle Regeneration Studies. A,B) Compiled maps of single tibialis anterior muscle sections 3 days after toxin injection showing examples of poor (A) and normal (B) toxin-induced injury. White boxed regions of each section map are shown as inset images for higher magnification viewing, Red, embryonic myosin heavy chain; green, collagen VI extracellular matrix protein; blue, DAPI nuclear stain. Scale bar, 100 μm. Please click here to view a larger version of this figure.
Table 1: Representative RNA Yield and Purity Measurements from Pooled Muscle cryosections. Sixteen tibialis anterior (TA) muscles were sectioned and pooled cryosection samples were processed for RNA. Purified RNA (1 μl) was analyzed with a nanospectrophotometer. Column statistics were performed in a spreadsheet, group comparisons were performed using statistical software.
To achieve best results with this method, keep embedding resin restricted to the lower third or half of the muscle during tissue cryopreservation because excess resin will slow the collection of the pooled cryosections and may increase embedding resin contamination in the RNA isolation. Also, careful attention during needle homogenization is important to maximize yield and minimize the probability of clogging the needle. The protocol may be modified by using a Luer-Lok syringe to protect against sample loss if the needle becomes blocked and requires high pressure to dislodge the clog. An additional needle homogenization step with a 25 or 26 gauge needle can also be added to produce a finer tissue suspension to further enhanced RNA yield. While chloroform could be substituted for BCP, this is not recommended as BCP is less toxic and results in lower levels of genomic DNA contamination in the aqueous phase during organic extraction of RNA12. Increasing the section thickness for pooled cryosections over 30 μm is also not recommended as homogenization will be less efficient.
If RNA yield is below desired levels, various strategies may be employed to increase recovery such as: i) increase the milligram quantity of starting material to increase possible yield; ii) reduce the section thickness below 30 μm to improve mechanical homogenization of the tissue; iii) increase the duration of sample incubation and needle homogenization in the organic extraction reagent to improve mechanical and chemical tissue disruption; and iv) if tissue chunks remain, perform a second extraction step with more rigorous needle homogenization. Alternately, there may be tissue-specific considerations, such as additional phase separation and precipitation steps for samples with high proteoglycan content13. During the RNA column purification, a larger elution volume can be used and performing a second elution can maximize total RNA recovery, but at the expense of RNA concentration. A post-column alcohol precipitation can be used to concentrate the RNA if low concentration is a concern with this modification. If RNA degradation is a problem, reducing time to cryopreservation during dissection, more rigorous cleaning of cryostat surfaces and tools to minimize RNase exposure, performing the needle homogenization step in a cold room, addition of an RNase inhibitor reagent to the cryosections14, and frequent replacement of RNase free solutions may each help to prevent or minimize exposure to RNases and reduce cleavage activity. It is possible that briefly bathing the tissue in an RNase inhibitor reagent after dissection, but before cryopreservation, may further reduce sample degradation. However, preliminary experiments should be performed to ensure that any such treatment does not increase ice crystals or other artifacts during cryopreservation.
While embryonic myosin heavy chain/collagen VI indirect immunofluorescence is used here as an example for muscle analysis of injury, thin cryosections mounted on microscope slides from these experiments can be used for any relevant histological stain that can be conducted on frozen sections, including immunofluorescent techniques with post-fixation and hematoxylin/eosin staining. Indeed, adaptations to the simple immunofluorescent protocol provided here may be necessary. For example, anti-mouse secondary antibodies used to detect a mouse primary antibody (e.g., eMHC) may also detect endogenous mouse immunoglobulins in the target tissue. Such endogenous antibodies typically accumulate in damaged or necrotic muscle fibers in injured or dystrophic muscle causing background immunofluorescent staining. A secondary control slide (with primary antibody omitted) should always be examined to assess the specificity of staining. If endogenous antibody background is problematic, pre-block steps should be added to the protocol to prevent or minimize detection of endogenous mouse immunoglobulins15.
The main limitations of the method are that it requires a cryostat and it is time consuming, which makes it relatively low throughput. For example, an expert in the technique was able to process up to 16 muscles for pooled cryosections and microscope slides (8 slides with duplicate sections of all 16 tissues) in approximately 9 to 10 hr. For novices to cryosectioning, collection of pooled cryosections from 2 to 4 samples could be reasonably mastered after cryostat training and one or two practice sessions, instead, obtaining quality cryosections for histology took more experience. Therefore, equipment, time, or training factors may make this method less useful for softer tissues that can be well homogenized with a manual pestle homogenizer.
In comparison with non-cryostat homogenization methods, striated muscle RNA preparations have been reported from muscle biopsies with RNA yields of 0.05 to 0.7 μg RNA pre mg of muscle16 and, more recently 0.27 to 1.08 μg RNA per mg of muscle17. Therefore, the technique described here provides RNA yields as good as or better than non-cryostat methods with the added advantage of enabling paired histological analyses from a contiguous region of the same sample. Notably, a previous study also used cryosectioning for homogenization in vertebral tissue and similarly found that cryosectioning tissue enhanced homogenization efficiency for RNA isolation13. When this technique was tested in bovine skeletal muscle samples, the average RNA yield per sample preparation was 4.09 ± 0.36 μg, at the low end of the normal range reported here13. Laser capture microdissection is another alternative for collection of tissue for RNA extraction from a cryosection. Laser capture is superior to this pooled cryosection method in that it allows the specificity to collect only a desired subset of cells from the section and it can be performed on a single tissue section up to 50 μm thick18. However, collection of a micro-dissected sample can be difficult and suitable equipment is not widely available, making pooled cryosection homogenization more accessible to researchers. When both methods are available, a preference to analyze a tissue sub-region for an application needing only small RNA quantities would favor laser capture microdissection while pooled cryosection homogenization is best when sub-region analysis is less important and higher quantities of RNA are needed.
While histological and RNA isolation methods are the focus here, the pooled cryosection method is easily adapted to prepare protein lysates for Western blot analyses or enzyme activity measurements. For example, pooled cryosections from the heart were solubilized for Western blot analyses4 and pooled cryosections from the TA were homogenized for succinate dehydrogenase activity assays of mitochondrial function5. Alternatively, genomic DNA and protein fractions can be separated from other phases during the organic extraction after RNA isolation, offering the potential to derive genomic DNA, protein, RNA, and histological measurements from a single tissue after a single cryostat session.
Overall, the main advantage of this method is to increase experimental flexibility by enabling multiple analytical approaches requiring different sample preparation from a single tissue. The method is most appropriate for muscle and other tissues with extensive intra- or extracellular structure that reduces the efficiency of pestle-based tissue homogenization.
The authors have nothing to disclose.
Madison Grant, Steven Foltz, Halie Zastre and Junna Luan provided technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number AR065077. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.
Cork | VWR Scientific | 23420-708 | Cut into small squares with a sharp blade. |
Plastic coverslip | Fisher Scientific | 12-547 | Used to orient the muscle during freezing. |
Low temperature thermometer | VWR Scientific | 89370-158 | |
2-methylbutane | Sigma | M32631-4L | Caution: hazardous chemical. Store in flammable cabinet. |
Embedding resin: "cryomatrix" | Thermo Fisher Scientific | 6769006 | Other embedding resins can be substituted for cryomatrix. |
Cryostat | Thermo Fisher Scientific | microm HM550 with disposable blade carrier | Any working cryostat should be sufficient for the protocol. |
Disposable cryostat blade | Thermo Fisher Scientific | 3052835 | Use an appropriate blade or knife for the cryostat to be used. |
RNAse decontamination solution: "RNase Zap" | Thermo Fisher Scientific | AM9780 | |
Analytical balance | Mettler Toledo | XS64 | |
Paint brush | Daler Rowney | 214900920 | Use to handle cryosections. Can be found with in stores with simple art supplies. |
Razor blade | VWR Scientific | 55411-050 | |
Microscope slide | VWR Scientific | 48311600 | |
RNA organic extraction reagent: TRIzol | Thermo Fisher Scientific | 15596026 | Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle. |
18 gauge needle | VWR Scientific | BD305185 | |
22 gauge needle | VWR Scientific | BD305155 | |
26 gauge needle | VWR Scientific | BD305115 | Optional. Can be used for a third round of sample trituration in the RNA extraction protocol. |
1 mL syringe | VWR Scientific | BD309659 | For very high value samples, a luer-lok syringe is recommended (e.g. VWR BD309628). |
1-bromo-3-chloropentane (BCP) | Sigma | B9673 | |
For 70% ethanol in DEPC water: 200 proof alcohol | Decon Laboratories, Inc. | +M18027161M | Mix 35 ml 200 proof alcohol + 15 mL DEPC water. |
For 70% ethanol in DEPC water: DEPC-treated water | Thermo Fisher Scientific | AM9922 | Mix 35 ml 200 proof alcohol + 15 mL DEPC water. |
RNA purification kit: PureLink RNA minikit | Thermo Fisher Scientific | 12183018A | Final steps of RNA preparation. |
DNase/Rnase-free water | Gibco | 10977 | DEPC-treated water can also be used. |
Spectrophotometer: Nanodrop 2000 | Thermo Fisher Scientific | NanoDrop 2000 | |
Dnase I | Thermo Fisher Scientific | AM2222 | Treat purified RNA to remove any DNA contamination before downstream appications. |
Hydrophobic pen | Thermo Fisher Scientific | 8899 | |
Dulbecco's PBS | Gibco | 14190 | PBS for immunofluorescence protocol. |
Donkey serum | Jackson ImmunoResearch Laoratories, Inc | 017-000-121 | Rehydrate normal donkey serum stock according to the manufacturer's instructions, then dilute an aliquot to 5% for immunofluorescence. Normal goat serum can also be used. |
eMHC antibody | University of Iowa Developmental Studies Hybridoma Bank | F1.652 | |
Collagen VI antibody | Fitzgerald Industries | #70R-CR009x | |
Donkey anti-rabbit AlexaFluor488 | Thermo Fisher Scientific | A21206 | |
Goat anti-mouse IgG1 AlexaFluor546 | Thermo Fisher Scientific | A21123 | |
DAPI (4',6-diamidino-2-phenylindole) | Thermo Fisher Scientific | D1306 | |
Aqueous mounting media: Permafluor | Thermo Fisher Scientific | TA-030-FM | Only use mounting media designed for fluorescent applications with anti-fade properties. |
Glass coverslip | VWR Scientific | 16004-314 | Use for mounting slides at the end of immunofluorescence protocl |
Image analysis software: ImageProExpress | Media Cybernetics, Inc. | Image-Pro Express, or more advanced products | Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process. E.g. ImageProPremier. |
Merge and map section images: Photoshop | Adobe | Photoshop | |
Cardiotoxin | Sigma | C9659 | Sigma C9659 has been discontinued. Other options for cardiotoxin are EMD Millipore #217503; American Custom Chemicals Corp. # BIO0000618; or Ge Script # RP17303; but these have not been validated. |
reverse transcription kit: Superscript III First-strand synthesis system | Thermo Fisher Scientific | 18080051 | Any validated, high quality reverse transcription reagents can be used. |
Standard PCR: GoTaq Flexi polymerase system | Promega | M8298 | Any validated, high quality Taq polymerase system can be used. If DNA sequencing is to be used for any application downstream of the PCR, then a high fidelity PCR system should be used instead. |
SYBR green | Thermo Fisher Scientific | S7585 | For use in qPCR when not using a dedicated qPCR master mix. Use with SuperROX (for Applied Biosystems instruments) and GoTaq Flexi polymerase and buffers. |
ROX: SuperROX, 15 mM | BioResearch Technologies, Inc. Novato CA | SR-1000-10 | SuperROX is more stable in the PCR reaction, so it is preferred for use as a qPCR passive reference dye over ROX (carboxy-X-rhodamine). For qPCR with Applied Biosystems instruments |
Real-time PCR | Applied Biosystems | 7900HT |